[烟草亚细胞定位]农杆菌瞬时侵染烟草-激光扫描共聚焦法

1、植物科学领域利用农杆菌侵染烟草进行体内瞬时表达的方法MethodfortransientexpressioninvivobyinfectingtobaccowithAgrobacterium一、原理(Principle)烟草叶片的瞬时表达系统常用来进行基因的亚细胞定位监测，监测蛋白在细胞中分布的位置，从而对其功能进行预测。通过基因翻译的蛋白与绿色荧光蛋白构成融合蛋白，在激光共聚焦仪器下观测绿色荧光的分布，判断蛋白表达的部位。同时，还可根据荧光的光强度检测蛋白的表达量。该过程是经根癌农杆菌介导的，进而将目的基因整合到到烟草的细胞内的。Tobaccoleaftransientexpressions

2、ystemsareoftenusedtomonitorthesubcellularlocalizationofgenes,monitorthedistributionofproteinsincells,andpredicttheirfunction.Thegenetranslatedproteinandgreenfluorescentproteinconstituteafusionprotein.Thedistributionofgreenfluorescenceisobservedunderalaserconfocalinstrumenttodeterminetheproteinexpres

3、sionsite.Atthesametime,theexpressionleveloftheproteincanalsobedetectedbasedonthelightintensityofthefluorescence.ThisprocessismediatedbyAgrobacteriumtumefaciens,whichintegratesthegeneofinterestintotobaccocells.二、材料与试剂1. 携带表达载体的农杆菌菌株(通常表达载体由35S启动子驱动)2. 2-4周的烟草植株3. LB培养基4. 乙酰丁香酮5. MES:2-(N-吗啉代)乙磺酸6. 抗生

4、素7. 注射器Materialsandreagents1. Agrobacteriumstraincarryinganexpressionvector(usuallytheexpressionvectorisdrivenbythe35Spromoter)2. Tobaccoplantsfor2-4weeks3. LBmedium4. Acetosyringone5. MES:2-(N-morpholino)ethanesulfonicacid6. Antibiotics7. syringe三、仪器1. 50ml离心管2. 光谱仪3. 紫外灯4. 荧光显微镜Third,theinstrument

5、1.50mlcentrifugetube2. spectrometer3. UVlamp4. fluorescencemicroscope四、步骤1. 挑取单克隆于5mlLB液体培养中，2830°震荡培养。通常，LB中加入100/ml庆大霉素(农杆菌株GV3101携带抗性)，50冯/ml大观霉素(载体携带)。2将1ml过夜培养的农杆菌转接到25mlLB液体培养基中(加有与1相同的抗生素，另外加入高压灭菌的乙酰丁香酮)。3. 检测过夜培养的菌液OD600的值。4. 5000g,15分钟集菌，用重悬液重悬菌体，最终ODeoo为0.4。5. 室温放置23h后注射烟草。6. 将侵染液装入5m

6、l注射器内，用拇指按压注射器反板将液体从叶片下表皮注射到烟草叶片内(勿使用子叶)。注射后，烟草叶片会出现湿润的现象。7. 注射后2-5天，在便携式长波长紫外灯下检测GFP荧光信号(只适用于荧光很强的叶片)。8. 通过荧光显微镜或者激光共聚交荧光显微镜检测GFP信号。同时，可以提取蛋白，检测蛋白的含量。Steps1. Pickasingleclonein5mlLBliquidcultureandshakecultureat2830C.Generally,LBisaddedwith100/mlgentamicin(agrobacteriumstrainGV3101carriesresistanee

7、)and50g/mlspectinomycin(carriedbycarrier).2. Transfer1mlofAgrobacteriumculturedovernightto25mlofLBliquidmedium(addthesameantibioticas1andaddautoclavedacetylsyringone).3. ChecktheOD600valueofthebacterialsolutionculturedovernight.4. 5000g,15minutestocollectbacteria,resuspendthebacteriawitharesuspensio

8、n,thefinalOD600is0.4.5. Injecttobaccoafterplacingatroomtemperaturefor23hours.6. Filltheinfectioussolutionintoa5mlsyringe,andpressthebackofthesyringewithyourthumbtoinjecttheliquidfromthelowerepidermisoftheleafintothetobaccoleaf(donotusecotyledons).Afterinjection,thetobaccoleaveswillappearmoist.7. 2-5

9、daysafterinjection,detecttheGFPfluoresceneesignalunderaportableIong-wavelengthUVlamp(onlyapplicabletohighlyfluorescentleaves).8. DetecttheGFPsignalusingafluoresceneemicroscopeoralasercopolymerizedfluoresceneemicroscope.Atthesametime,proteincanbeextractedandthecontentofproteincanbedetected.五、配方1. 加有相

10、应抗生素的LB液体培养基(一种抗生素是菌株携带，一种为载体携带)。2. 乙酰丁香酮(100mM溶于乙醇，-20C储存)。3. 1MMgCl24. 重悬液(10mMMgCl2,10mM2-(N-吗啉代)乙磺酸(pH5.6)高温高压灭菌15分钟，100uM乙酰丁香酮，高温高压灭菌)。Five,formula1. LBliquidmediumsupplementedwithcorrespondingantibiotics(oneantibioticiscarriedbythestrainandoneiscarriedbythecarrier).2. Acetylsyringone(100mMinetha